Background Rheumatoid arthritis (RA) patients frequently produce autoantibodies against citrullinated proteins, proteins in which an arginine residue is converted into a citrulline by peptidylarginine deiminase (PAD). In view of the putative pathophysiological role of the immune response to citrullinated proteins, it is important to obtain a comprehensive view of the citrullinated proteins present in the inflamed joints of RA patients. To facilitate these studies, new methods for the detection of citrullinated proteins are needed, particularly because the antibodies required for the classical anti-modified citrulline methodology cannot be produced anymore. Monoclonal antibodies to citrullinated proteins have been developed previously and some of these display a wide reactivity with citrullinated proteins, although some bias for the citrulline in a certain amino acid context exists.
Objectives To generate new reagents and procedures for studying citrullinated proteins in biological samples and to identify citrullinated proteins present in the synovial fluid of RA patients.
Methods To generate new tools for the detection of citrullinated proteins DNA aptamers were selected from a library containing 40 degenerate deoxyribonucleotides by the SELEX procedure using a peptide containing a (modified) citrulline residue flanked by 6 glycines on either side as target. In an alternative procedure a phenylglyoxal derivative, which under highly acidic conditions reacts with the ureido group of citrulline, was used. An azido group was attached to phenylglyoxal to facilitate the visualization of the peptidylcitrulline-phenylglyoxal reaction products by a click chemistry approach, using an alkyne-containing probe. Citrullinated proteins present in the synovial fluid of RA patients were characterized by western blotting and mass spectrometry.
Results For the detection of citrullinated proteins, DNA aptamers targeting either peptidylcitrulline or peptidylcitrulline with a chemically modified ureido group were generated. Their molecular features and target specificities are currently being studied and will be presented. The consecutive reactions of azido-phenylglyoxal with citrullinated proteins and of biotinylated azadibenzocyclooctyne or bicyclononyne with the product of the first reaction allow the detection of citrullinated proteins on western blots. The reaction conditions have been optimized to obtain the highest sensitivity and the lowest background. The analysis of citrullinated proteins from the synovial fluid of RA patients showed clear differences between individual patients and led to the identification of 53 citrullinated proteins. Three of the newly identified citrullinated proteins, apolipoprotein E, myeloid nuclear differentiation antigen and β-actin, were studied in more detail. All of these appeared to contain citrullinated epitopes targeted by anti-citrullinated protein antibodies from RA patient sera.
Conclusions Both aptamer-mediated and click chemistry-based technologies represent promising new technologies for the characterization of citrullinated proteins. Synovial fluids of RA patients contain a wide variety of citrullinated proteins. Apolipoprotein E, myeloid nuclear differentiation antigen and β-actin are identified as novel citrullinated antigens in the RA synovial fluid.
Disclosure of Interest None Declared
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